Human thymic stromal lymphopoietin (hTSLP) (GenBank accession number: NM—033035), an interleukin-7 (IL-7) like cytokine, which is produced by human epithelial stroma and mast cells, initiates the allergic response by the stimulation of dendritic cells (DC). The deduced 159-amino acid protein is 43% identical to mouse TSLP, contains a 28-residue signal sequence, 6 cysteines, and 2 putative N-glycosylation sites. Native Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis showed expression of a 23 kilo Dalton (kDa) protein, whereas the calculated molecular mass of the mature protein is 14.9 kDa, suggesting that hTSLP is glycosylated. hTSLP contains 7 basic C-terminal amino acids (aa) N-Lysine-Lysine-Arginine-Arginine-Lysine-Arginine-Lysine-C (KKRRKRK) (SEQ ID NO:115) and 6 cysteines probably involved in disulfide bond formation.
hTSLP is highly expressed by epithelial cells of inflamed tonsils and keratinocytes of atopic dermatitis and its expression is associated with Langerhans cell migration and activation2.
The TSLP receptor complex is a heterodimer comprised of the TSLP receptor (TSLPR) and IL-7 receptor alpha (IL-7Ra) chain. The receptor is expressed primarily on monocytes and myeloid-derived DC, as well as on B lymphocytes3.
Allergy is the result of a complex immune cascade leading to the dysregulated production of the Thymus-derived helper cell type 2 (Th2) subset lymphocyte cytokines, the generation of allergen-specific IgE-producing B lymphocytes and the subsequent activation and degranulation of mast cells upon allergen challenge.
DC play an important role in several models of allergy whereby TSLP-activated human DC produce Th2-attracting chemokines but no IL-112, and induce naïve CD4- and CD8-antigen-positive T lymphocyte differentiation into effector cells with a typical pro-allergic phenotype.
Atopic dermatitis (AD) represents a chronic, relapsing inflammatory skin disease with characteristic clinical features4. Genetic background, environmental exposures such as food allergens, aeroallergens, microbial antigens, or stress, and distinct immunological predispositions all contribute to the development of periodic, itchy eczematous skin lesions in afflicted patients. Several soluble factors have been shown to be increased in the peripheral blood of patients with AD. These cytokines and chemokines play an important role in regulating DC differentiation, activation and migration and are important in coordinating the trafficking of immune cells. hTSLP, which is produced by human epithelial stroma and mast cells, initiates the allergic response by the stimulation of DC. TSLP activated DC produce the CC chemokines that induce the chemotaxis and polarization of allergen-specific effector lymphocytes5. Thus, epithelial- and stromal-cell-derived TSLP might represent one of the factors initiating the allergic responses, and could be a target for a curative therapeutic approach to allergy.
In recent studies, one anti-human TSLP polyclonal antibody was described (R&D Systems, AF1398). This antibody was produced in sheep immunized with purified E. coli-derived recombinant TSLP. Human TSLP specific sheep IgG was purified by hTSLP affinity chromatography. This polyclonal antibody was selected for its ability to neutralize hTSLP bioactivity and showed less than 1% cross-reactivity with recombinant murine TSLP. The Neutralization Dose50 (ND50) for this antibody was defined as that concentration of antibody required to yield one-half maximal inhibition of the recombinant hTSLP activity on the responsive cell line mouse BaF/3 cells co-transfected with IL-7Ra and hTSLPR chains, as an assay. The ND50 was determined to be approximately 0.05-0.25 μg/ml in the presence of 0.5 ng/ml recombinant hTSLP. The disadvantage of polyclonal antibodies is that they are in a limited supply as there is a restricted supply of serum from the same treated animal. In addition, polyclonal antibodies recognize multiple epitopes on the same antigen and may have undesired cross-reactivity. While polyclonal serum contains a mixture of both high and low affinity binders, targeting also a range of epitopes, a monoclonal antibody approach make sure to select the most useful candidate for a therapeutic use.
Animal-derived polyclonal antibodies when injected in humans constitute a foreign protein in a human host, they often elicit an antiglobulin response due to their immunogenicity in human. This antiglobulin response, which is predominantly directed against the constant domains of the animal antibodies, usually precludes treatment after repeated administration.